Apparatus and methods for investigating the binding of analytes in solution to a receptor are known. One such apparatus (and its associated computer control and data-processing means) called BIAcore (BIAcore is a trademark of Pharmacia Biosensor AB; BIA stands for biospecific interaction analysis) has been devised, which uses the phenomenon of surface plasmon resonance (SPR) to study the binding of analytes to receptors immobilised on a sensor chip. The apparatus and theoretical background are fully described in the literature (e.g. Jonsson et al., 1991, BioTechniques 11, 620-627). Essentially, the technique involves the immobilisation of a receptor to the special surface of a sensor chip, contacting the chip with a flow of sample containing the analyte of interest, and then measuring the change in the surface optical characteristics of the sensor chip arising from the binding of the analyte of interest.
The specific nature of the antibody/antigen interaction means that such reactions are particularly amenable to investigation using the BIAcore system. For example Jonsson et al. (1991, cited above) disclose the use of high affinity antibodies for concentration determinations. In this system the sensor chip required regeneration with HCl after each assay. There are several other reported studies which describe, for example, the interaction of synthetic peptides with immobilised monoclonal antibodies (Altschuh et al., 1992, Biochemistry 31, 6298-6304), and binding reactions between immobilised monoclonals and HIV core protein p24 and vice versa; Karlsson et al., 1991, Journal of Immunological Methods 145, 229-240).
Sensors for applications such as on-line monitoring of chromatography, fermentation or even in vivo use, should ideally give rapid, reversible and sensitive responses to changes in concentrations of proteins or other biomolecules. Normally antibodies have strong binding of antigen and can only be used as monitor of the concentration as a dosimeter. For such applications regeneration of the antibody (to recreate vacant antigen-binding sites), or the use of disposable items is required. However, in many environments regeneration with chemical agents can not be done.
It is apparent from the prior art that for an antibody-based solid-phase biosensor to be capable of measuring reversibly (without the need for regeneration), the antibody should have a high dissociation rate constant in binding antigen ("k off") (Anderson & Miller, 1988, Clinical Chemistry 34, 1417-1421) but those authors do not disclose antibodies or fragments thereof having intrinsically high dissociation rate constants for continuous on-line sensing.
However, there are considerable difficulties in actually obtaining receptor molecules (such as antibodies) with the desired properties. This is because virtually all screening methods for searching for suitable candidate molecules require a washing step. Such a washing step would tend to remove the very molecules possessing the properties for which one was screening.
Thus, all of the prior art disclosures regarding biosensors relate to the use of "mature" monoclonal antibodies produced as a result of the primary immunisation and subsequent boosting of the relevant laboratory animal (typically, a mouse). These antibodies therefore bind antigen too strongly for the reaction to be readily reversible. Moreover, these antibodies are typically directed against haptens. The shortest reversible response time reported (Anderson & Miller, above) was in the range 5-15 minutes (for the comparatively high analyte concentration range of 5-100 mM), which is not quick enough to follow parameters for many "on-line/real-time" applications. Further, the apparatus disclosed therein required the use of dialysis tubing. This results in severe limitations on the molecular weight of analytes that may be studied.